Thermal unfolding of dodecameric manganese.glutamine synthetase (MW=622,000) at pH 7 and approximately 0.02 ionic strength occurs in two observable steps: a small reversible transition (Tm approximately 42 degreesC; delta H = 0.2 cal/g) followed by a large irreversible transition (Tm approximately 81 degreesC; delta H = 5.6 cal/g) in which secondary structure is lost and soluble aggregates form. Secondary structure, hydrophobicity, and oligomeric structure of the equilibrium intermediate are the same as for the native protein whereas some aromatic residues are more exposed. Urea (3 M) destabilizes the dodecamer (with a tertiary structure similar to that without urea at 55 degreesC) and inhibits aggregation accompanying unfolding at less than or equal to 0.2 mg protein/ml. With increasing temperature (30 - 70 degreesC) or incubation times at 25 degreesC (5 - 35 h) in 3 M urea, only dodecamer and unfolded monomer are detected. In addition, the loss in enzyme secondary structure is pseudo-first-order with a half-time of 1030 s at 20.0 degreesC in 4.5 M urea. Differential scanning calorimetry of the enzyme in 3 M urea shows one endotherm (Tmax approximately 64 degreesC; deltaH = 4.1 +/- 0.5 cal/g). The enthalpy change for dissociation and unfolding agrees with that determined by urea titrations by isothermal calorimetry (delta H = 14 +/- 4 cal/g; Zolkiewski M, Nosworthy NJ, Ginsburg A. 1995. Protein Sci 4:1544-1552), after correcting for the binding of urea to protein sites exposed during unfolding (~ 10 cal/g). Refolding and assembly to active enzyme occurs upon dilution of urea after thermal unfolding. Thus, it is the redistribution of urea to unfolded monomer sites that promotes the concerted (apparently irreversible) dissociation of dodecameric glutamine synthetase to unfolded monomers without detectable intermediates.